Abstract

Anti-HER2 monoclonal antibody trastuzumab is used as treatment in patients with HER2 positive breast cancer. However, resistance inevitably occurs for all responders if trastuzumab is given as monotherapy. Although trastuzumab was developed to block HER2 signaling, several studies have shown that trastuzumab does not decrease HER2 phosphorylation. In a recent study, we have shown that in BT474 and SKBR3 cells HER2 phosphorylation is maintained by activation of the other HER receptors via their dimerization with HER2. The activation of alternative HER receptors is due to an increase of HER ligand release, which is mediated by a negative feedback loop involving PKB and ADAM17.

In this study, we investigated the effect of trastuzumab treatment in vivo. Our hypothesis was that the described PKB feedback loop could explain differences in response to trastuzumab in vivo. Resistant xenograft samples may have higher levels of ADAM17 and HER ligands compared to responding xenograft samples, and thus maintaining their HER2 signaling.

To study this hypothesis, BT474 xenografts were treated with five doses of trastuzumab or were left to grow untreated. The xenograft samples were then paraffin-embedded and analysed by immunohistochemisty. We scored the samples for HER2 membrane and cytoplasmic levels using an Intensity Percentage Scoring (IPS) system. Although, membrane HER2 levels were significantly reduced after trastuzumab treatment, there was a lot of heterogeneity between samples. We hypothesized that differences in drug deliver could be an explanation for this heterogeneity, so we co-stained for HER2 and CD31 (a blood vessel marker). Ten different parts of one sections were then independently scored for HER2 levels and blood vessel count. A lower membrane HER2 staining was found to correlate with a higher number of blood vessels. This finding suggests that HER2 membrane levels are downregulated when trastuzumab is delivered to the tissue. Drug delivery is therefore very important for an effective response to trastuzumab treatment.

In line with our previously published data, staining for ADAM17, HER ligand heregulin, phospho-EGFR and phospho-HER3 receptors was increased in trastuzumab treated xenografts compared to untreated samples. Interestingly, in our preliminary results not all of the trastuzumab treated samples showed this increase, indicating a possible difference in response to treatment. In future, comparing xenograft size to the staining intensity of these proteins should give us an understanding of the difference in response.

These in vivo findings are in line with our in vitro findings and offer treatment opportunities for overcoming resistance in patients. We propose that trastuzumab should be combined with a panHER inhibitor or an ADAM inhibitor to overcome the acquired drug resistance for HER2 positive breast cancer patients. A similar strategy could help overcome resistance to other therapies targeting this pathway